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Biomedical subjects

R K Mishra

Publications and source records attributed to R K Mishra.

At least 55 records · Page 3Linked to original sources
PubMed1996

Targeting nucleic acid secondary structures by antisense oligonucleotides designed through in vitro selection.

Using an in vitro selection approach, we have isolated oligonucleotides that can bind to a DNA hairpin structure. Complex formation of these oligonucleotides with the target hairpin involves some type of triple-stranded structure with noncanonical interaction, as indicated by bandshift assays and footprinting studies. The selected oligomers can block restriction endonuclease cleavage of the target hairpin in a sequence-specific manner. We demonstrate that in vitro selection can extend the antisense approach to functional targeting of secondary structure motifs. This could provide a basis for interfering with regulatory processes mediated by a variety of nucleic acid structures.

Binding Sites↗
PubMed1996

Modulation of brain catecholamine absorbing proteins by dopaminergic agents.

Catecholamine absorbing proteins (CATNAPs) are localized in the brain and thus far have no known biochemical and pharmacological characteristics consistent with other receptor proteins or metabolic enzymes in the central nervous system. The oxidative metabolism of catecholamines in the brain, especially the catabolism of dopamine and its conjugation with metabolic brain proteins, results in the production of highly toxic free radicals. Since such processes are implicated in the pathophysiology of various neurodegenerative diseases, including parkinsonism, and since CATNAPs bind catecholamines with high affinity, there is a need to further investigate if these novel proteins could play a protective role against these harmful catecholamine metabolites. In this study, we demonstrate the purification, pharmacological characterization and modulation of CATNAPs, as the first steps necessary to elucidate the function of these proteins in the brain. First, CATNAPs were identified from tissues using [3H]N-n-propylnorapomorphine (a specific dopamine receptor agonist) and [125I]6-hydroxy-5-iodo[N(N-2,4-dinitro-phenyl)- aminopropyl]1,2,3,4-tetrahydronaphthalene ([125I]DATN; a highly specific ligand synthesized in our laboratory). Three proteins, with molecular masses of 47, 40 and 26 kDa, were identified and purified, which allowed for the subsequent production of antibodies against each of these CATNAPs. The effects of in vivo chronic administration of several dopaminergic agents on CATNAPs were also examined by Western immunoblotting. L-3,4-Dihydroxyphenylalanine (L-DOPA) treatment in rats resulted in the increase of all of the three proteins, as compared to controls. Treatment in rats with the dopamine depleting agent, reserpine, produced a significant decrease in all of the three CATNAPs. In addition, the effects of direct administration of apomorphine, dopamine, epinephrine, isopropylnorepinephrine, norepinephrine, N-n-propylnorapomorphine and 6-hydroxydopamine on CATNAP levels in rats were examined. Interestingly, we observed an increase (as compared to control) of the 47, 40 and 26 kDa proteins in animals treated with dopamine, norepinephrine, N-n-propylnorapomorphine and apomorphine. In contrast, animals treated with 6-hydroxydopamine showed significant decreases in the levels of all three proteins. It is evident that as the concentration of catecholamines increases, there is a corresponding increase in the levels of CATNAPs in the brain. These results clearly demonstrate the pharmacological modulation of CATNAPs by dopaminergic agents and suggest their possible role in the cytoprotection against damage caused by free radicals generated by oxidative stress.

Adrenergic Uptake Inhibitors↗
PubMed1996

Spectrophotometric determination of thorium in standard samples and monazite sands based on the floated complex of thorium with N-hydroxy-N,N'-diphenylbenzamidine and thorin.

A selective and sensitive spectrophotometric method for the determination of Th(IV) has been based on the reaction with thorin and subsequent extraction of the red-orange coloured complex with N-hydroxy-N,N'-diphenylbenzamidine (HDPBA) in benzene as floated complex at pH 2.2. The complex in ethanol exhibits a maximum absorbance at 495 nm, with a molar absorptivity of 6.0x10(4) l mol(-1) cm(-1), with a Sandell's sensitivity of 3.9x10(-3) microg cm(-2). The method follows Beer's law up to 3.0 microg Th(IV) ml(-1). None of the common cations and anions tested interfere. The detection limit of the method is 0.04 microg Th(IV) ml(-1), the RSD (n=10) is 1.4%. The method has been successfully employed for the determination of thorium in various standard and monazite samples.

Journal Article↗
PubMed1996

Interaction of NMDA and dopamine D2L receptors in human neuroblastoma SH-SY5Y cells.

To understand the mechanism of interaction of the dopamine D2L receptors with NMDA receptors, we have developed a model by transfecting human neuroblastoma SH-SY5Y cells with the human dopamine D2L receptor gene. In vitro blockade of NMDA receptors by the specific antagonists MK-801 and (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) on human neuroblastoma SH-SY5Y cells expressing human dopamine D2L receptors resulted in a significant increase in the density of D2L receptors without a significant change in receptor affinity. Moreover, the dopamine receptor mRNA level increased by approximately 50% by the blockade of NMDA with MK-801. These results suggest a possible interaction of NMDA and dopamine D2L receptors in neuroblastoma SH-SY5Y cells. This system would serve as an excellent model to study the molecular mechanisms involved in the interaction of these two receptors.

Base Sequence↗
PubMed1996

A neurochemical basis for the antipsychotic activity of loxapine: interactions with dopamine D1, D2, D4 and serotonin 5-HT2 receptor subtypes.

Loxapine is a typical neuroleptic that shows great structural and functional homology to the atypical antipsychotic clozapine. Chronic loxapine treatment is usually associated with extrapyramidal symptoms (EPS), whereas clozapine treatment is not. Conversely, loxapine does not produce the agranulocytosis that often results from protracted clozapine treatment. Earlier studies of loxapine have usually implicated D2 receptor blockade as the cause of the tardive dyskinesia that occurs with chronic treatment. More recently, loxapine's ability to potentiate serotonergic neurotransmission has also been implicated. In this study, the pharmacological affinities of loxapine for the dopamine D1, D2, D4, as well as serotonin-2 (5-HT2) and NMDA receptor subtypes, were investigated through direct radioreceptor assays. The findings indicate that loxapine displays an extremely strong binding affinity for dopamine D4 and serotonin 5-HT2 receptors, which suggests that both serotonergic and dopaminergic mechanisms contribute to the antipsychotic drug action and EPS associated with loxapine in the treatment of schizophrenia.

Antipsychotic Agents↗
PubMed1995

Ontogenic development of dopamine D4 receptor in rat brain.

The postnatal development of rat brain dopamine D4 receptor gene expression was studied in animals 1 day to 1 year old, using the polymerase chain reaction technique. The level of expression of the D4 mRNA was appreciable at birth (day 1), increased to maximum at day 3, and showed declines at day 28. D4 mRNA expression remained unchanged at the ages of 6 months to 1 year. The mRNA expression at day 1 is about 50% of that observed on day 3, and declines approximately 50% by day 28. In contrast, the dopamine D2 mRNA expression was maximum at day 28, which is consistent with the reported studies.

Animals↗
PubMed1995

Improved leishmanicidal effect of phosphorotioate antisense oligonucleotides by LDL-mediated delivery.

We have designed antisense oligonucleotides that can interact with lipoproteins in order to use them as vectors to facilitate the uptake by those cells expressing the corresponding receptor. Phosphorothioate (PS) oligonucleotides were linked at the 5' end to a palmityl group giving rise to PSPal conjugates. Such a modification enables the oligonucleotide to form a stable non-covalent complex with low density lipoproteins (LDL) through hydrophobic interactions. The antisense effect of LDL-oligonucleotide complexes was assayed by targeting the mini-exon sequence of Leishmania amazonensis in infected mouse peritoneal macrophages. A 16-mer antisense PSPal oligonucleotide/LDL complex exerted a more pronounced sequence-specific effect than the free oligomer: about 25% and 10% of infected macrophages were cured by a 48 h incubation in the presence of 2.5 microM of the complexed and the free oligomer, respectively. When oxidized LDL was used instead of the native one for complexation, a further 2-fold increase in the antisense effect was observed suggesting that alternative (unregulated) scavenger receptor can be used for more efficient delivery of antisense oligonucleotides into macrophages. In addition, a significant reduction of the parasitic load was observed in those cells that were not fully cured.

Animals↗
PubMed1995

Modulation of N-methyl-D-aspartate (NMDA) antagonist-induced darting behaviour by the peptidomimetic PAMTA.

The N-Methyl-D-Aspartate (NMDA) receptor has attracted much attention in recent years due to its involvement in both the functions and dysfunctions of CNS neurotransmission. The existence of multiple sites by which NMDA receptor channel function can be pharmacologically modified and the interaction between glutamate and other neurotransmitter systems such as dopamine, provide exciting therapeutic avenues for related CNS disorders. In the present study, a novel synthetic analogue of the endogenous brain peptide L-prolyl-L-leucyl glycinamide (PLG) has demonstrated a significant modulatory action on the NMDA receptor. On the basis of radioligand binding studies, the novel synthetic peptide 5-[1(S)-(2(S)-pyrrolidinylcarbonyl)amino-3-methylbutyl]-2- tetrazolylacetamide (PAMTA) has been suggested to act at a polyamine site on the NMDA receptor complex. Scatchard analysis of [3H]MK-801 binding revealed that in the presence of 100 microM PAMTA, a single binding site was obtained with the Kd being increased from 2.5 +/- 0.2 nM to 6.2 +/- 0.1 nM. The ability of PAMTA to inhibit the binding of [3H]MK-801 was sensitive to the presence of both spermidine (polyamine agonist) and arcaine (polyamine antagonist). Analyses of the binding profiles of various NMDA receptor antagonists support PAMTA's interaction with the polyamine site on this receptor complex. Furthermore, we have investigated the behavioural profile of the peptidomimetic PAMTA, by studying its effect on stereotypic behaviours induced by the NMDA receptor antagonist, CPP (3(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid). Male Sprague-Dawley rats cannulated bilaterally into the medial prefrontal cortex were injected with PAMTA, CPP, a CPP/PAMTA combination, or a saline control.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Covalent affinity labeling of brain catecholamine-absorbing proteins using a high-specific-activity substituted tetrahydronaphthalene.

The recently described catecholamine-absorbing proteins (CATNAPs) are expressed within the CNS and have been shown to participate in neurochemical processes involving dopamine and several structurally related catecholamines. Specifically, CATNAPs have been implicated in participating directly in oxidative mechanisms involving reactive species (such as free radicals) derived from these compounds. Toxic free radicals generated from endogenous catecholamines have been identified as a major cause of neuronal tissue injury and are implicated in several disease processes. CATNAPs were first identified by their ability to react covalently with tritiated dopaminergic compounds, incorporating low levels of radioactivity under appropriate reaction conditions. The biochemical characterization of CATNAPs has until now been hampered by the lack of a suitable high-specific-activity probe to allow the rapid detection of these proteins. We describe here the synthesis and labeling characteristics of a high-specific-activity substituted tetrahydronaphthalene derivative (6-hydroxy-[125I]iodo-[N-(N-2',4'- dinitrophenyl)aminopropyl]-2-amino-1,2,3,4-tetrahydronaphthalene), which covalently incorporates into CATNAPs with the same tissue distribution, molecular weight patterns, and pharmacology as observed for the previously studied tritiated catecholamines. This compound greatly enhances the detection of CATNAPs and will facilitate further biochemical characterization of these proteins.

Absorption↗
PubMed1995

An experiment on cognitive remediation of word-reading difficulty.

Cognitive remediation of decoding deficit was attempted by following a theoretically based program. The theory identifies four major cognitive processes: Planning, Attention, Simultaneous, and Successive (PASS) processing. The PASS Remedial Program (PREP) provides 10 structured tasks that are aimed at developing internalized strategies for mainly successive processes (6 tasks) and simultaneous process (4 tasks); deficits in either of the two may lead to poor decoding. Through its "global process" training and curriculum-related "bridging" training, PREP facilitates application of internalized strategies arrived at inductively for learning word decoding and spelling; it does not provide direct teaching of rules or exercises. To test the efficacy of PREP, we divided 51 children with decoding difficulties in Grade 4 into two groups: PREP (both global and bridging) and no treatment. In the second part of the study, children from the no-treatment group received either the global or the bridging part of PREP. The relative efficacy of training was tested by pre-, and posttests of performance on a standard word-decoding test (the WRMT-R), as well as on some cognitive tests (e.g., the CAS). The largest improvement in word decoding occurred for the PREP combined global and bridging treatment. The mechanism through which PREP improves word reading is discussed, as is the use of PREP for children at risk of developing dyslexia.

Child↗
PubMed1994

Design, synthesis, X-ray analysis, and dopamine receptor-modulating activity of mimics of the "C5" hydrogen-bonded conformation in the peptidomimetic 2-oxo-3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-1- pyrrolidineacetamide.

3(R)-(7a(S)-Hexahydro-1-oxo-3,3-dimethyl-1H-pyrrolo[1,2-c]imidazol-2-yl) - oxo-1-pyrrolidine-acetamide (2) and 3(R)-[1-(2,5-dioxopyrrolidino[3,4-c]piperazino)]-2-oxo-1- pyrrolidineacetamide (3) were designed and prepared as mimics of the "C5" hydrogen-bonded structure found in the crystal structure of 2-oxo-3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-1- pyrrolidineacetamide (1). Both compounds effectively restrict the psi 1 torsional angle to very near the value found in the X-ray structure of 1 as seen in the X-ray crystallographic determination of 2 and methyl 3(R)-[1-(2,5-dioxopyrrolidino[3,4-c]piperazino)]-2-oxo-1- pyrrolidineacetate (11), a diketopiperazine intermediate in the synthesis of 3. These analogs were tested for their ability to enhance the binding of the dopamine D2 receptor agonist N-propylnorapomorphine (NPA) in the absence and presence of 5'-guanylylimidodiphosphate (Gpp(NH)p). Both compounds enhanced [3H]-NPA binding in a dose-dependent manner by increasing both the binding affinity of the agonist and the number of high-affinity sites available for binding. Both 2 and 3 also attenuated the Gpp(NH)p-induced conversion of D2 receptor high-affinity states to the low-affinity states.

Amino Acid Sequence↗
PubMed1994

Antisense phosphorothioate oligonucleotides: selective killing of the intracellular parasite Leishmania amazonensis.

We targeted the mini-exon sequence, present at the 5' end of every mRNA of the protozoan parasite Leishmania amazonensis, by phosphorothioate oligonucleotides. A complementary 16-mer (16PS) was able to kill amastigotes--the intracellular stage of the parasite--in murine macrophages in culture. After 24 hr of incubation with 10 microM 16PS, about 30% infected macrophages were cured. The oligomer 16PS acted through antisense hybridization in a sequence-dependent way; no effect on parasites was observed with noncomplementary phosphorothioate oligonucleotides. The antisense oligonucleotide 16PS was a selective killer of the protozoans without any detrimental effect to the host macrophage. Using 16PS linked to a palmitate chain, which enabled it to complex with low density lipoproteins, improved the leishmanicidal efficiency on intracellular amastigotes, probably due to increased endocytosis. Phosphorothioate oligonucleotides complementary to the intron part of the mini-exon pre-RNA were also effective, suggesting that antisense oligomers could prevent trans-splicing in these parasites.

Animals↗
PubMed1994

Striatal G-proteins: effects of unilateral 6-hydroxydopamine lesions.

To investigate the molecular mechanisms of denervation-induced dopamine receptor supersensitivity, levels of striatal G-proteins were examined in 6-hydroxydopamine (6-OHDA)-lesioned rats. Increased levels of the alpha-subunits of Gs and G(olf), as measured by immunoblotting, were observed 8 days postlesion and remained elevated up to day 28 (25-45% over the time course). G(i) levels were significantly increased only on day 8 (20%) and G(o) levels remained unaltered. These results suggest that stimulatory G-proteins may be involved in the long-term maintenance of dopamine receptor supersensitivity following denervation.

Animals↗
PubMed1994

Differential influence of DNA supercoiling on in vivo strength of promoters varying in structure and organisation in E. coli.

DNA supercoiling is known to influence promoter activity in vitro and in vivo in a promoter-dependent manner in prokaryotes. In order to investigate how topology may influence promoter function, we have studied two kinds of promoter variants, (i) where only the spacer region is altered, and (ii) where the same promoter is tandemly repeated in either the same or opposite orientation. These promoters respond very differently to alterations in DNA supercoiling, suggesting that the overall structure of the promoter and its context contribute to the differential response to alterations in supercoiling in vivo.

Base Sequence↗
PubMed1994

Distribution of cholecystokinin receptors in the bovine brain: a quantitative autoradiographic study.

Quantitative in vitro receptor autoradiography was used to study the distribution of cholecystokin receptors in the bovine brain. [125I]Bolton-Hunter cholecystokinin octapeptide binding was described in whole hemisphere sagittal and coronal sections using cholecystokinin octapeptide, devazepide and L-365,260 as competitors to identify the subtypes. High levels of cholecystokinin receptors were found in the cortex, where they presented a laminar distribution which varied from area to area. The basal ganglia, the caudate nucleus, nucleus accumbens and putamen presented high to moderate levels of cholecystokinin binding, whereas only very low labelling was found in the globus pallidus. Cholecystokinin binding was present in all portions of the bovine hippocampus; high levels were found in the dentate gyrus, CA1 subfield of Ammon's horn, subiculum and presubiculum. Moderate to high levels were also found in the amygdala, inferior colliculus and olfactory tract, while most of the hypothalamic and thalamic nuclei exhibited very low or no cholecystokinin binding. Low cholecystokinin binding was uniformly distributed across cell layers of the bovine cerebellar cortex. Competition of [125I]Bolton-Hunter cholecystokinin octapeptide binding in the cortex, nucleus accumbens, caudate nucleus, hippocampus, cerebellum and brainstem was much greater in the presence of L-365,260 than devazepide, thereby suggesting that the majority of cholecystokinin receptors in these regions are of the cholecystokinin-B subtype. The results of this study, when compared to distribution profiles in other mammalian species, provide further evidence for species differences in the distribution of cholecystokinin receptors in the brain. The results also support the possible interaction between cholecystokinin and dopaminergic systems in areas of the brain containing dopaminergic terminals, such as the frontal cortex, nucleus accumbens, caudate-putamen and olfactory tubercle.

Animals↗
PubMed1994

The role of speech processes and memory in reading disability.

The performance of poor and average readers on cognitive tasks comprising naming time, speech rate, and word series recall and on a phonemic segmentation task was examined in the first part of this study by computing relationships among the tasks and their relation to reading. In the second part of this study, we investigated the performance of the poor and average readers in detail on naming time by analyzing the audiotaped protocols on a computer equipped with a sound system and an analysis program. The subjects were third- and fourth-grade students. Results showed that naming time and speech rate were dependable measures of phonological coding skills and were better predictors of word decoding than were phonemic segmentation and word recall. Poor readers were slower in each of the six rows of the naming-time task, and they paused longer between the rows. A need for studying the development of the cognitive skills along with word decoding is discussed.

Child↗
PubMed1994

Cognitive patterns of children with dyslexia: a comparison between groups with high and average nonverbal intelligence.

We tested the hypothesis that children with dyslexia identified by word decoding deficit will be poor in specific cognitive processes that require successive processing and rapid articulation, irrespective of their high or average nonverbal IQ. Children with dyslexia and normally achieving children between 9 years and 11 years of age were divided into four groups comprising average-IQ and high-IQ children with dyslexia and normal readers. All children were administered measures of planning, attention-arousal, simultaneous and successive processes, phonemic segmentation, and nonverbal IQ. Results confirmed the hypothesis: The cognitive tasks that differentiated children with dyslexia from nondyslexic children irrespective of IQ were the successive tasks, as well as two tasks of attention that required articulation and/or phonological coding. Tasks that demanded both phonological coding and articulation correctly classified children with dyslexia and nondyslexic children up to 80%. The importance of remedial training appropriate for alleviating the cognitive and word decoding deficits is discussed.

Analysis of Variance↗